Role of p53 Missense Mutations on Tumorigenesis in Vivo

项目摘要 癌症是一组复杂的、不同种类的和毁灭性的疾病；然而，一个共同的主题是 肿瘤抑制基因TP53的复发和高频突变。与其他肿瘤不同 抑制子，大多数TP53改变是错义突变，这些表现为功能获得表型 依赖于转录重新连接。这项建议的重点是p53错义突变在 三阴性乳腺癌(TNBC)乳腺癌是一种分布广泛的恶性肿瘤，占所有癌症的15%。 全球女性癌症死亡人数。80%-88%的基底细胞样乳腺癌，其中大多数是TNBC， 肿瘤抑制基因TP53的突变。我们最近开发出了第一只基因工程小鼠 P53错义突变所致体细胞乳腺癌模型的建立。此条件模型表示一个主要的 随着其表达来自内源性位点的突变型P53并在间质中维持野生型P53和 免疫系统。此外，我们的模型忠实地模仿了人类TNBC的组织学，并用 一年的延迟。此外，在这些小鼠中，使用CRISPR-Cas9缺失突变的p53会导致肿瘤 退化和存活率增加，表明乳腺肿瘤对突变型p53上瘾。此外，我们 见肿瘤消退时T淋巴细胞浸润增加。在这项研究中，我们建议理解 由体细胞P53基因突变启动的TNBC的生物学和确定这些肿瘤成瘾的原因 突变型p53。这项工作将我们优雅的、与疾病相关的TNBC小鼠模型与最新的 ART基因组图谱全面了解突变型P53驱动乳房的机制 癌症。此外，我们将使用最近从我们的模型中衍生出来的细胞系，这些细胞系也对突变的p53上瘾 作为一个平台，进行功能基因组学筛查，以识别这些癌症中的新脆弱性。 基于免疫的疗法使癌症治疗发生了革命性的变化。因此，要有效地推进突变型p53的定向 治疗方面，需要对免疫状况和反应有全面的了解。因为我们的 体细胞乳房模型独特地保留了WT-P53微环境和免疫隔间，我们处于 理想的位置，以检查对突变型p53定向治疗的免疫反应。我们的具体目标是：目标 1.为了了解推动P53基因缺失和突变小鼠乳腺癌发生的潜在生物学因素， 我们将进行完整的外显子序列分析。确定导致成瘾的分子途径 对于突变型p53乳腺癌，我们将对体细胞性p53R172H乳腺癌进行RNA-SEQ分析。 不删除突变型P53。目的2.确定突变型P53下游的分子通路 对于使用CRISPR/Cas9功能筛查的肿瘤维持是必不可少的。目标3.理解和开发 乳腺癌患者对突变型P53基因治疗抑制的免疫反应。因为我们的条件鼠标 模型与人类散发性肿瘤的潜在分子病理学相同，它将更好地预测 人类对药物的反应，因此是临床前测试的宝贵工具。

Project Summary Cancer is a collection of complex, heterogeneous, and devastating diseases; however, one common theme is recurrent and high-frequency mutations in the TP53 tumor suppressor gene. In contrast to other tumor suppressors, most TP53 alterations are missense mutations and these exhibit gain-of-function phenotypes that are dependent of transcriptional rewiring. This proposal is focused on the role of p53 missense mutations in triple negative breast cancer (TNBC). Breast cancers are widespread malignancies representing 15% of all cancer deaths in women worldwide. 80-88% of basal-like breast cancers, the majority of which are TNBC, have mutations in the TP53 tumor suppressor. We have recently developed the first genetically engineered mouse model of somatic breast cancer driven by p53 missense mutations. This conditional model represents a major advance as it expresses mutant p53 from the endogenous locus and maintains wild-type p53 in the stroma and immune system. Moreover, our model faithfully mimics the histology of human TNBC, and develops TNBC with a one year latency. Moreover, in these mice, deletion of mutant p53 with CRISPR-Cas9 results in tumor regression and increased survival, indicating that the breast tumors are addicted to mutant p53. In addition, we see increased infiltration of T lymphocytes upon tumor regression. In this study, we propose to understand the biology of TNBC initiated by a somatic p53 driver mutation and determine why these tumors are addicted to mutant p53. This work combines our elegant and disease relevant mouse models of TNBC with state-of-the- art genomic profiling to comprehensively understand the mechanisms underlying mutant p53 driven breast cancer. Moreover, we will use cell lines recently derived from our model that are also addicted to mutant p53 as a platform to undertake a functional genomics screen to identify novel vulnerabilities in these cancers. Immune based therapies have revolutionized cancer care. Therefore to effectively advance mutant p53 directed therapies, a comprehensive understanding of the immune landscape and response is needed. Since our somatic breast model uniquely retains a WT p53 microenvironment and immune compartment, we are in an ideal position to examine the immune response to mutant p53 directed therapies. Our specific aims are: Aim 1. To understand the underlying biology that drives breast cancer development in p53-null versus mutant mice, we will perform whole exome sequence analyses. To identify the molecular pathways that lead to addiction of mutant p53 breast cancers, we will perform RNA-seq analyses of somatic p53R172H breast tumors with and without deletion of mutant p53. Aim 2. To determine the molecular pathways downstream of mutant p53 that are essential for tumor maintenance using CRISPR/Cas9 functional screens. Aim 3. To understand and exploit the immune response to therapeutic inhibition of mutant p53 in breast cancer. Because our conditional mouse model shares the underlying molecular pathology with human sporadic tumors, it will be more predictive of human responses to drugs, and thus a valuable tool in preclinical testing.